There are known a serum diagnosis of identifying a tumor marker in blood serum, a tissue diagnosis by biopsy, and a cytodiagnosis, as examples of the cancer diagnoses currently practiced at clinical places.
The tumor marker is a substance such as a protein, a glycoprotein, or a lipid, which is a product in a tumor cell as a result of expression of a gene that is no longer expressed in a process of generation or growth of an individual organism. The serum diagnosis is an assessment on a disease stage or malignancy grade of a tumor by detection of the tumor marker. Most of the currently available tumor markers, however, do not have high cancer specificity. Also, since the expression level of the tumor marker is very low in an early stage cancer, diagnosis accuracy is relatively low. For the above reasons, in the current medical institutes, reliability on cancer diagnosis using the tumor marker is not so high.
The tissue diagnosis and the cytodiagnosis are diagnostic methods, wherein after staining tissue or cell specimens obtained by biopsy, pathologists and cell screeners make an assessment on the stained specimens based on the conditions or stained patterns of the specimens through microscopic observation, based on their own medical or clinical experiences. The staining methods are different among the medical institutes. Also, the final assessment depends on medical or clinical experiences of pathologists or cell screeners. Accordingly, it is difficult to make a definite diagnosis in a delicate stage such as a moderately differentiated stage or stage 1. In a worse case, selection on medication treatment may be erred, thereby leading to serious progression of a cancer.
In view of the above, the TNM classification, as a criterion for standardizing the diagnostic methods, has been established, and globally been spread. The TNM classification for a cancer is a criterion adopted by the Union International Contre Cancer (UICC), and shows progress levels of malignant tumors. “T” represents the size of a primary tumor, “N” represents the level of a lymph node metastasis, and “M” represents a distant metastasis. “T” is classified from level “1” indicating that the tumor is localized within the tissue to level “4” indicating that tumorigenesis appears in a site other than the affected tissue. “N” is classified from level “0” indicating that no local lymph node metastasis is observed to level “3” indicating that a lymph node metastasis is histopathologically observed. “M” is classified from level “0” indicating that no distant metastasis is observed to level “1” that a distant metastasis is observed. In any of the parameters of “T”, “N”, and “M”, the larger the numerical value is, the poorer the prognosis is, which means that the malignancy grade of the cancer is high.
The TMN (sic, correctly TNM) classification is generally used because it is useful for determination of a treatment method, or prognosis assessment. However, even if patients are diagnosed to be in an early stage of a cancer according to the TNM classification, in the case of a breast cancer, relapse by a distant metastasis is observed in about 10 to 20% of the patients within 5 years after the diagnosis, leading to death, which is a clinically important matter. Although the assessment based on the TNM classification is useful for medical staffs to grasp clinical conditions of patients at the time of diagnosis, the assessment has not successfully led to accurate prediction on a prognosis.
In addition to the above diagnostic method, there is proposed a method of diagnosing malignancy grade of a cancer by measuring the DNA content of cancer cells, using a fluorescence-activated cell sorter (FACS), and based on the measurement result, in light of a point that a cancer tissue contains a large amount of polyploids such as triploids or tetraploids. However, it is required to disperse an obtained biopsy specimen into single cells in order to measure the biopsy specimen by the FACS. It is generally not easy to disperse a biopsy specimen into single cells, and a high level skill is required to do this. For the above reasons, application of the cancer diagnostic method using the FACS in the current medical institutes is reportedly difficult.
Under the foregoing circumstances, there is a need of establishing a diagnostic method capable of providing a definite diagnosis without the need of a cumbersome preparation of samples, namely, dispersion into single cells, and with less variation in diagnosis resulting from individual assessments or different assessment methods in medical institutes at clinical places.
In recent years, there is expected a molecular diagnosis using an apparatus, as a standardized cancer diagnostic method with less variation in diagnosis by diagnosticians.
One of the molecular diagnoses is a method of assessing malignancy grade of cancer, using a DNA chip, based on a comparison result on expression of gene transcript between a biopsy specimen and a standard specimen. However, the expression of gene transcript does not have a high correlation to produced proteins in a living organism. Accordingly, effectiveness of the diagnostic method is thought to be less trustworthy in the clinical places.
Development and study have been progressed concerning a molecular diagnosis based on a protein which is expressed in a living organism. For instance, D1 proposes a diagnostic method, wherein expression levels of CDK1 and CDK4 in a sample, and mutation of p53 according to needs, are used as indicators. D2 proposes a diagnostic method of a cancer and precancerous states, using overexpression of CDK4, CDK6, or cyclin dependent kinase inhibitor (CDK inhibitor).
It is known that expression of cyclin dependent kinase (CDK) is high in a cell where proliferation is induced by a growth factor. Generally, however, a certain amount of CDK exists in a cell, and the CDK shows its activity in a specific stage of a cell cycle depending on the kind of CDK, by binding to cyclin molecules under activation of phosphorylation or a like action, and forming a complex of CDK and cyclin. Also, it is known that the CDK inhibitor is bound to CDK and/or a complex of cyclin and CDK, thereby inhibiting the CDK activity. As mentioned above, since the actual cell cycle is complicatedly controlled, the assessment simply based on the expression level of CDK, cyclin, or CDK inhibitor does not provide a sufficient indicator for a controlled state of the cell cycle.
D3 discloses a diagnostic method of using CDK activities as an indicator for assessing malignancy grade of cancer, considering binding to cyclin, or influence of an inhibitor, and a method of measuring CDK activities without using a radioactive substance. However, data concerning the CDK activities to be measured by the disclosed method fails to provide diagnosis precision sufficient as a substitute of the cancer diagnoses currently practiced at the clinical places.
It is sometimes required to know sensitivity of tumor cells in vitro to an anticancer agent, as an indicator on effectiveness of a chemotherapy using the anticancer agent. Examples of the known in vitro tests of measuring sensitivity of tumor cells to agents such as an anticancer agent are MTT assay method and DISC method. The MTT assay method is a method, utilizing a phenomenon that MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) can be cleaved by a reductase (succinate dehydrogenase) in mitochondria when the MTT is trapped in a living cell, but that the MTT cannot be cleaved when trapped in a dead cell. The MTT assay method comprises cultivating a cancer cell for 3 hours by addition of the MTT and an agent to be measured, measuring light absorbency i.e. succinate dehydrogenase activity, and quantitatively determining the number of living cells. It is reported that 80% of assessment prediction results by the in vitro assessment method accorded with relapse or no relapse by an actual chemotherapy using an anticancer agent, and that 68% of prediction positive i.e. sensitive to the anticancer agent by the in vitro assessment method showed no relapse by the actual chemotherapy using the anticancer agent (see D4).
DISC method is a method comprising contacting a cancer cell with an anticancer agent for 4 days, staining the dead cells with a fast green, identifying the living cells by staining the living cells with hematoxylin-eosin to assess whether the cancer cell is dead or alive. It is reported that a probability i.e. a matching rate between in vitro results and actual results is 82.7%, and a probability on positive prediction rate is 62.5% (see D4).
In any of the above methods, the positive prediction rate is less than 70%, which is an insufficient indicator for determining whether a chemotherapy using an anticancer agent is to be started.    D1: JP patent publication No. 2002-504683    D2: JP patent publication No. 2002-519681    D3: JP patent publication No. 2002-335997    D4: Weisenthal, L. M. and Nygren. P (2002) Current status of cell culture drug resistance testing(CCDRT), http://weisenthal.org/oncol_t.htm